1. Papillomavirus infection and viral gene expression. Human papillomavirus type 16 (HPV16) or 18 (HPV18) infection, acquired primarily via sexual transmission, is widely recognized as a leading cause of cervical and anal cancer. Infection with oncogenic HPV in other tissues could also lead to development of cancer. For example, we recently demonstrated that colorectal oncogenic HPV infection is common in patients (51%) with colorectal cancer. High prevalence and incidence of cervical HPV infection has been observed among HIV-positive and immunodeficient women. Cervical cancer has been the most common malignancy among women with AIDS in both Europe and the United States. Two viral oncoproteins, E6 and E7, of HPV16 and HPV18 are involved in cervical carcinogenesis and are known to destabilize cellular tumor suppressor proteins p53 and pRb, respectively. Recently, we showed that high-risk HPV infection also deregulates the expression of tumor-suppressive miR-34a and p18Ink4c through viral oncoprotein E6. In HPV16 and HPV18, E6 and E7 are transcribed as a single bicistronic RNA bearing 3 exons and 2 introns, with the intron 1 in the E6 coding region. Splicing of the intron 1 in the E6E7 bicistronic pre-mRNAs is highly efficient and the majority of the spliced transcripts in cancer tissues and cervical cancer cell lines are E6*I, a spliced product without intron 1. We have hypothesized that the E6 is expressed from a small portion of the bicistronic RNAs with retention of the intron 1. This hypothesis raises several important questions: 1. Why is an efficient splicing of the intron 1 in the E6E7 pre-mRNAs needed since the splicing disrupts E6 expression? 2. What proportion of the E6E7 pre-mRNAs escape the splicing of intron 1 and what regulates this escaping? 3. How could an RNA molecule containing an intron be exported to the cytoplasm to translate E6 protein? To address these questions will lead us to understand the mechanisms that are involved in RNA splicing regulation. In the past few years, we demonstrated that the intron 1 splicing subjects to exon definition (size) by a cap structure on the RNA 5' end. Although the E6E7 mRNAs with the intron 1 retention encode viral oncoprotein E6, the spliced E6*I mRNAs is mainly resposible for for the expression of E7 oncoprotein. Based on these observations, we have developed several E6 and E7-specific siRNAs to selectively silence the expression of each viral oncogene. We further demonstrated that aberrant expression of oncogenic and tumor-suppressive microRNAs in cervical cancer is required for cancer cell growth and viral oncoproteins are partially responsible for this deregulation. 2. KSHV Gene expression and post-transcriptional regulation. KSHV is a lymphotropic DNA tumor virus that induces Kaposi sarcoma (KS), primary effusion lymphoma (PEL) or body cavity-based B-cell lymphoma, and multicentric Castleman disease. Among those malignancies, KS occurs frequently in patients infected with HIV. Latent KSHV infection in KS lesions and PEL-derived B cells features the highly restricted expression of only five viral genes. The lytic KSHV infection can be induced by chemicals or hypoxia in PEL-derived B cells with latent KSHV infection. In this lytic switch, a KSHV transactivator, ORF50, is absolutely required. ORF50 is an immediate-early gene transcribed as a polycistronic RNA with 5 exons and 4 introns. ORF50 is positioned in the virus genome with both K8 (an early gene encoding a K-bZIP protein) and K8.1 (a late gene encoding a viral envelope glycoprotein) and shares a single polyadenylation site downstream of K8.1 coding region. Accordingly, the transcripts of the three genes overlap each other and undergo extensive RNA splicing. Our initial study was to investigate how this complicated gene expression is regulated. We found there are two major isoforms of spliced RNA products, alpha (exclusion of K8 intron 2 or ORF50 intron 3) and beta (inclusion of K8 intron 2 or ORF50 intron 3) in KSHV lytic infection. By profiling the transcription and splicing products of ORF50, K8 and K8.1, we demonstrated that KSHV K8beta is derived from a splicing intermediate and antagonizes K8alpha-mediated induction of p21 and p53 and blocks K8alpha-CDK2 interaction. We further showed that KSHV ORF57 promotes RNA splicing of K8beta to produce K8alpha. KSHV ORF57 is a ICP27 homolog of herpes simplex viruses and is essential for KSHV replication and virus production. Demonstration of that ORF57, a mRNA transcript accumulator (MTA), is a viral splicing factor was a surprise since its homologs in other herpesvirus are all splicing suppressive in reported studies. ORF57 is a phosphorylated nuclear protein bearing three nuclear localization signals in its N-terminus and its activities mostly take place in the nucleus of an infected cell. In the analysis of the gene structure and expression of KSHV ORF56 (viral primase), ORF57, ORF58 (EB virus BMRF2 homology) and ORF59 (viral DNA polymerase processing factor), we demonstrated that both ORF56 and ORF59 are expressed as bicistronic RNAs that subject to ORF57 up-regulation. Works are in progress to understand the mechanisms on how KSHV ORF57 regulates the expression of viral and cellular genes post-transcriptionally.